The Systematic Evolution of Ligands by EXponential enrichment method, or SELEX, is a combinatorial chemistry technique for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to one or more target ligands. The method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve a desired level of binding affinity and selectivity. SELEX has been used to evolve nucleic acid aptamers of extremely high binding affinity to a variety of targets. Some of these targets include, for example, lysozyme (Potty et al.), thrombin (Long et al.), human immunodeficiency virus trans-acting responsive element (HIV TAR) (Darfeuille et al.), hemin (Liu et al.), interferon gamma (Min et al.), vascular endothelial growth factor (VEGF) (Ng et al.), prostate specific antigen (PSA) (Savory et al.; Jeong et al.).
Aptamers have found applications in many areas, such as biotechnology, medicine, pharmacology, microbiology, and analytical chemistry, including chromatographic separation and biosensors.
Interestingly, structure-switching aptamers or SwAps have also found multiple applications. A review of SwAps as biosensors was published in 2009 by Sefah et al. Changes in fluorescence intensities between free and bound aptmamer complexes have been described to detect cocaine by aptamer-based capillary zone electrophoresis (Deng et al.). Multiple small molecule analytes have been detected by a similar method (Zhu et al.) High surface area, solid phase sol-gel-derived macroporous silica films have also been shown to be suitable platforms for high-density affinity-based immobilization of functional single stranded-aptamer molecules, allowing for binding of both large and small target ligands through SwAps with robust signal development (Carrasquilla et al.)
Aptamers have further been used for protein and small molecule purification using affinity chromatography. Indeed, aptamer affinity chromatography has been applied to protein purification (Romig et al.) and in the separation of mature dendritic cells from immature dendritic cells (Berezovski et al.). However, aptamer affinity chromatography has not to the inventors' knowledge been shown in the prior art to apply to the purification of cells, viruses or antibodies.
A major problem encountered when dealing with aptamer-based affinity chromatography to purify target ligands such as viruses, cells and certain other biological materials is the need for elevated temperatures or the addition of detergents to alter the conformation of the SwAp and to subsequently allow the release of the captured biomolecular target ligand from the solid medium or chromatography column. These harsh regeneration techniques decrease significantly the viability of cells and viruses, denature proteins and irreversibly change the structure of biomolecules. Furthermore, the lack of an efficient regeneration technique that can be generalized to other target-specific aptamers has been a challenge to the widespread use of aptamers for purification. Concerns have also been raised with regard to the possible cross-reaction between aptamers and other contaminants that might exist in the mixture containing the biomolecule to be purified. As such, until now, these problems have made the utilization of aptamers for the purification and recovery of purified targets such as viruses and cells very difficult to achieve.
The methods currently available for purification of viruses include: differential centrifugation, size exclusion chromatography (SEC) and heparin affinity column chromatography. These techniques are not without challenges. Sucrose differential gradient centrifugation is conventional for virus isolation in small quantities, but it is difficult to scale-up, is labour-intensive and requires long processing times, which may decrease the infectivity of viruses (Diallo et al.) SEC does not separate well from cell debris or large molecular aggregates with similar sized viruses, and is followed with additional concentration steps such as ultrafiltration or polyethylene glycol-6000 precipitation. The heparin column purification utilizes sepharose beads conjugated to linear anionic heparin molecules. This technique is used to purify proteins containing a heparin-binding domain as well as retroviruses. Although, this heparin method yields a purer product than the density gradient method, it still requires additional SEC purification from cationic proteins and salt.